The enormous complexity of biological samples (e.g., from proteomics) and the need for both biological and technical analysis replicates impose major challenges for multidimensional separation platforms in regard to both sensitivity and sample throughput. A major potential attraction of the Ion Mobility Spectrometry-Time-Of-Flight-Mass Spectrometry (IMS-TOF MS) platform is the ability to provide separation speeds exceeding that of conventional condense phase separations by orders of magnitude. Known limitations of most typical IMS-TOF MS platforms that impede this attraction include the need for extensive signal averaging due to factors that include significant ion losses in the IMS-TOF interface and an ion utilization efficiency of less than ˜1% with continuous ion sources (e.g., ESI).
A multiplexed IMS-TOF approach has been shown to provide up to 10-fold increase in sensitivity as compared to the conventional signal averaging approach in regard to analysis of peptide mixtures. This sensitivity improvement is based on introduction of multiple ion packets into an IMS drift tube on the time scale of a single measurement in the signal averaging experiment. Each ion packet injection occurs during a constant IMS gate open event. Ion injection process is governed by an extended pseudo-random sequence that mitigates diffusion-driven ion cloud expansion and enables efficient ion accumulation prior to each gate open event. Short (˜100 us) IMS gate open events minimize contribution of the ion injection term on IMS resolving power. A complete description of this invention is found in pending U.S. patent application Ser. No. 11/701,752, entitled “Method of Multiplexed Analysis Using Ion Mobility Spectrometer” the contents of which are hereby incorporated by reference in its entirety.
The need in multiplexing the IMS-TOF is strongly dictated by the total number of analyte molecules delivered to the ion trap (preceding the IMS drift tube) per unit time and by the charge capacity of that trap. Given lower abundance signals, ion trap may remain under filled with ions in the course of IMS separation, implying no need in multiplexing to attain efficient ion utilization. In this case, ion accumulation over the entire IMS separation would be rather beneficial for achieving high sensitivity. For higher abundance ion signals, the ion trap will be over filled with ions in a fraction of IMS separation timescale, thus requiring the purging the trap multiple times throughout a single IMS separation. Therefore, a combination of approaches is needed to maximize instrument sensitivity in analysis of complex samples with broad dynamic range.
The present invention describes an approach for increasing the dynamic range of a multidimensional IMS-TOF system in analysis of biological samples. The key feature of this invention is that the multidimensional system automatically adjusts to analyte abundances in the course of experiments, providing an ultra-high sensitivity for a variety of biological samples that significantly vary in complexity and dynamic range.
Additional advantages and novel features of the present invention will be set forth as follows and will be readily apparent from the descriptions and demonstrations set forth herein. Accordingly, the following descriptions of the present invention should be seen as illustrative of the invention and not as limiting in any way.